Reagents test strip adapted for receiving an unmeasured sample while in use in an apparatus

ABSTRACT

A method for determining the presence of an analyte in a fluid is described along with various components of an apparatus specifically designed to carry out the method. The method involves taking a reflectance reading from one surface of an inert porous matrix impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to another surface and migrates through the matrix to the surface being read. Reflectance measurements are made at two separate wavelengths in order to eliminate interferences, and a timing circuit is triggered by an initial decrease in reflectance by the wetting of the surface whose reflectance is being measured by the fluid which passes through the inert matrix. Repeatability is insured by a normalization technique performed on the light source before each reading, and an alignment method operated on the reagent strip prior to emplacement on the apparatus. The method and apparatus are particularly suitable for the measurement of glucose levels in blood without requiring separation of red blood cells from serum or plasma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of Ser. No. 09/784,993 filed Feb. 15,2001, now U.S. Pat. No. 6,489,133 which is a Division of Ser. No.09/323,442 filed May 28, 1999, now U.S. Pat. No. 6,268,162B1 issued Jul.31, 2001, which is a Continuation of Ser. No. 08/965,745 filed Nov. 7,1997, now U.S. Pat. No. 5,968,760 issued Oct. 19, 1999, which is aContinuation of Ser. No. 08/941,868 filed Sep. 30, 1997, now U.S. Pat.No. 5,843,692 issued Dec. 1, 1998, which is a Continuation of Ser. No.08/691,154 filed Aug. 1, 1996, now abandoned, which is a Continuation ofSer. No. 08/408,064 filed Mar. 21, 1995, now U.S. Pat. No. 5,563,042issued Oct. 8, 1996, which is a Continuation of Ser. No. 08/148,055filed Nov. 5, 1993, now U.S. Pat. No. 5,426,032 issued Jun. 20, 1995,which is a Division of Ser. No. 08/006,859 filed Jan. 21, 1993, nowabandoned, which is a Division of Ser. No. 07/819,431 filed Jan. 10,1992, now abandoned, which is a Division of Ser. No. 07/187,602 filedApr. 28, 1988, now U.S. Pat. No. 5,179,005 issued Jan. 12, 1993, whichis a Continuation-in-part of Ser. No. 06/896,418 filed Aug. 13, 1986,now U.S. Pat. No. 4,935,346 issued Jun. 19, 1990.

FIELD OF THE INVENTION

The present invention relates to a test device and method for thecolorimetric determination of chemical and biochemical components(analytes) in aqueous fluids, particularly whole blood. In one preferredembodiment it concerns a test device and method for colorimetricallymeasuring the concentration of glucose in whole blood.

BACKGROUND OF THE INVENTION

The quantification of chemical and biochemical components in coloredaqueous fluids, in particular colored biological fluids such as wholeblood and urine and biological fluid derivatives such as blood serum andblood plasma, is of ever-increasing importance. Important applicationsexist in medical diagnosis and treatment and in the quantification ofexposure to therapeutic drugs, intoxicants, hazardous chemicals and thelike. In some instances, the amounts of materials being determined areeither so miniscule—in the range of a microgram or less per deciliter—orso difficult to precisely determine that the apparatus employed iscomplicated and useful only to skilled laboratory personnel. In thiscase the results are generally not available for some hours or daysafter sampling. In other instances, there is often an emphasis on theability of lay operators to perform the test routinely, quickly andreproducibly outside a laboratory setting with rapid or immediateinformation display.

One common medical test is the measurement of blood glucose levels bydiabetics. Current teaching counsels diabetic patients to measure theirblood glucose level from two to seven times a day depending on thenature and severity of their individual cases. Based on the observedpattern in the measured glucose levels, the patient and physiciantogether make adjustments in diet, exercise and insulin intake to bettermanage the disease. Clearly, this information should be available to thepatient immediately.

Currently a method widely used in the United States employs a testarticle of the type described in U.S. Pat. No. 3,298,789 issued Jan. 17,1967 to Mast. In this method a sample of fresh, whole blood (typically20-40 μl) is placed on an ethylcellulose-coated reagent pad containingan enzyme system having glucose oxidase and peroxidase activity. Theenzyme system reacts with glucose and releases hydrogen peroxide. Thepad also contains an indicator which reacts with the hydrogen peroxidein the presence of peroxidase to give a color proportional in intensityto the sample's glucose level.

Another popular blood glucose test method employs similar chemistry butin place of the ethylcellulose-coated pad employs a water-resistant filmthrough which the enzymes and indicator are dispersed. This type ofsystem is disclosed in U.S. Pat. No. 3,630,957 issued Dec. 28, 1971 toRey et al.

In both cases the sample is allowed to remain in contact with thereagent pad for a specified time (typically one minute). Then in thefirst case the blood sample is washed off with a stream of water whilein the second case it is wiped off the film. The reagent pad or film isthen blotted dry and evaluated. The evaluation is made either bycomparing color generated with a color chart or by placing the pad orfilm in a diffuse reflectance instrument to read a color intensityvalue.

While the above methods have been used in glucose monitoring for years,they do have certain limitations. The sample size required is ratherlarge for a finger stick test and is difficult to achieve for somepeople whose capillary blood does not express readily.

In addition, these methods share a limitation with other simplelay-operator colorimetric determinations in that their result is basedon an absolute color reading which is in turn related to the absoluteextent of reaction between the sample and the test reagents. The factthat the sample must be washed or wiped off the reagent pad after thetimed reaction interval requires that the user be ready at the end ofthe timed interval and wipe or apply a wash stream at the required time.The fact that the reaction is stopped by removing the sample leads tosome uncertainty in the result, especially in the hands of the homeuser. Overwashing can give low results and underwashing can give highresults.

Another problem that often exists in simple lay-operator colorimetricdeterminations is the necessity for initiating a timing sequence whenblood is applied to a reagent pad. A user will typically have conducteda finger stick to obtain a blood sample and will then be required tosimultaneously apply the blood from the finger to a reagent pad whileinitiating a timing circuit with his or her other hand, therebyrequiring the use of both hands simultaneously. This is particularlydifficult since it is often necessary to insure that the timing circuitis started only when blood is applied to the reagent pad. All of theprior art methods require additional manipulations or additionalcircuitry to achieve this result. Accordingly, simplification of thisaspect of reflectance reading instruments is desirable.

The presence of red blood cells or other colored components ofteninterferes with the measurements of these absolute values, therebycalling for exclusion of red blood cells in these two prior methods asthey are most widely practiced. In the device of U.S. Pat. No. 3,298,789an ethyl cellulose membrane prevents red blood cells from entering thereagent pad. Similarly, the water-resistant film of U.S. Pat. No.3,630,957 prevents red blood cells from entering the pad. In both casesthe rinse or wipe also acts to remove these potentially interfering redblood cells prior to measurement.

Accordingly, there remains a need for a system of detecting analytes incolored liquids, such as blood, that does not require removal of excessliquid from a reflectance strip from which a reflectance reading isbeing obtained.

SUMMARY OF THE INVENTION

Novel methods, compositions and apparatus are provided for diagnosticassays comprising a hydrophilic porous matrix containing a signalproducing system and a reflectance measuring apparatus which isactivated upon a change in reflectance of the matrix when fluidpenetrates the matrix. The method comprises adding the sample, typicallywhole blood, to the matrix which filters out large particles, such asred blood cells, typically with the matrix present in the apparatus. Thesignal-producing system produces a product which further changes thereflectance of the matrix, which change can be related to the presenceof an analyte in a sample.

Exemplary of the diagnostic assay system is the determination of glucosein the whole blood, where the determination is made without interferencefrom the blood and without a complicated protocol subject to use error.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention can be more readily understood by reference to thefollowing detailed description when read in conjunction with theattached drawings, wherein:

FIG. 1 is a perspective view of one embodiment of a test devicecontaining the reaction pad to which the fluid being analyzed isapplied;

FIG. 2 is a block diagram schematic of an apparatus that can be employedin the practice of the invention;

FIG. 3 is a perspective view of a preferred embodiment of the testdevice of the present invention emplaced within a measuring system;

FIG. 4 is an enlarged pan view of a preferred embodiment of the testdevice of the present invention emplaced within a measuring system;

FIG. 5 is a graph plotting a second order correction to eliminate errorsdue to chromatography effects during the use of the present invention;

FIGS. 6 a and 6 b are scattergrams of glucose values as measured by apreferred embodiment of the present invention (called the singlewavelength MPX system) plotted against Yellow Springs Instruments (YSI)glucose values; and

FIGS. 7 a, 7 b, 7 c and 7 d are scattergrams of glucose values asmeasured by a second preferred embodiment of the present invention(called the double wavelength MPX system) plotted against Yellow SpringsInstruments (YSI) glucose values.

DETAILED DESCRIPTION OF THE INVENTION

The Reagent Element

The subject invention provides an improved rapid and simple methodologyemploying reliable and easy to operate apparatus for the determinationof analytes such as glucose, particularly involving an enzyme substratewhich results in the production of hydrogen peroxide as an enzymeproduct. The method involves applying to a porous matrix a small volumeof whole blood, sufficient to saturate the matrix. It is to be notedthat the present system is capable of determining glucose levels fromoptical readings of whole blood samples. Separation of plasma from bloodin the sample is unnecessary, and the present invention avoids therequirement of this step. In addition, this system is capable ofperforming accurate readings as long as only a small volume saturatesthe matrix of the text strip. Above this threshold, the reading isvolume independent.

Bound to the matrix are one or more reagents of a signal producingsystem, which results in the production of a product resulting in aninitial change in the amount of reflectance of the matrix. The matrix istypically present in a reflectance-measuring apparatus when blood isapplied. The liquid sample penetrates the matrix, resulting in aninitial change in reflectance at the measurement surface. A reading isthen taken at one or more times after the initial change in reflectanceto relate the further change in reflectance at the measurement surfaceor in the matrix as a result of formation of the reaction product to theamount of analyte in the sample.

For measurements in blood, particularly glucose measurements, wholeblood is typically used as the assay medium. The matrix contains anoxidase enzyme which produces hydrogen peroxide. Also contained in thematrix will be a second enzyme, particularly a peroxidase, and a dyesystem which produces a light-absorbing product in conjunction with theperoxidase. The light-absorbing product changes the reflectance signalof the matrix system. With whole blood, readings are taken at twodifferent wavelengths, with the reading at one wavelength used tosubtract out background interference caused by hematocrit, bloodoxygenation, and other variables which may affect the result. Thus, thepresent invention is capable of analyzing samples of whole blood.

A reagent element is employed which comprises the matrix and the membersof the signal producing system contained within the matrix. The reagentelement may include other components for particular applications. Themethod requires applying a small volume of blood, which typically hasnot been subject to prior treatment (other than optional treatment withan anticoagulant), to the matrix. Timing of the measurement is activatedor initialized by the apparatus' automatically detecting a change inreflectance of the matrix when fluid penetrates the matrix. The changein reflectance over a predetermined time period as a result of formationof reaction product is then related to the amount of analyte in asample. The intensity of the light source used to analyze the sample is,of course, also carefully monitored and regulated, to insure therepeatability of the measurement.

The first component of the present invention to be considered is areagent element, conveniently in the shape of a pad, comprising an inertporous matrix and the component or components of a signal-producingsystem, which system is capable of reacting with an analyte to produce alight-absorbing reaction product, impregnated into the pores of theporous matrix. The signal-producing system does not significantly impedethe flow of liquid through the matrix.

In order to assist in reading reflectance, it is preferred that thematrix have at least one side which is substantially smooth and flat.Typically, the matrix will be formed into a thin sheet with at least onesmooth, flat side. In use, the liquid sample being analyzed is appliedto one side of the sheet whereby any assay compound present passesthrough the reagent element by means of capillary, wicking, gravity flowand/or diffusion actions. The components of the signal producing systempresent in the matrix will react to give a light absorbing reactionproduct. Incident light impinges upon the reagent element at a locationother than the location to which the sample is applied. Light is thusreflected from the surface of the element as diffuse reflected light.This diffuse light is collected and measured, for example by thedetector of a reflectance spectrophotometer. The amount of reflectedlight will be related to the amount of analyte in the sample, usuallybeing an inverse function of the amount of analyte in the sample.

The Matrix

Each of the components necessary for producing the reagent element willbe described in turn. The first component is the matrix itself.

The matrix will be a hydrophilic porous matrix to which reagents may becovalently or noncovalently bound. The matrix will allow for the flow ofan aqueous medium through the matrix. It will also allow for binding ofprotein compositions to the matrix without significantly adverselyaffecting the biological activity of the protein, e.g., enzymaticactivity of an enzyme. To the extent that proteins are to be covalentlybound, the matrix will have active sites for covalent bonding or may beactivated by means known to the art. The composition of the matrix willbe reflective and will be of sufficient thickness to permit theformation of a light-absorbing dye in the void volume or on the surfaceto substantially affect the reflectance from the matrix. The matrix maybe of a uniform composition or a coating on a substrate providing thenecessary structure and physical properties.

The matrix will usually not deform on wetting, thus retaining itsoriginal conformation and size. The matrix will have a definedabsorbance, so that the volume which is absorbed can be calibratedwithin reasonable limits, variations usually being maintained belowabout 50% preferably not greater than 10%. The matrix will havesufficient wet strength to allow for routine manufacture. The matrixwill permit non-covalently bound reagents to be relatively uniformlydistributed on the surface of the matrix.

An exemplary of matrix surfaces are polyamides, particularly withsamples involving whole blood. The polyamides are convenientlycondensation polymers of monomers of from 4 to 8 carbon atoms, where themonomers are lactams or combinations of diamines and di-carboxylicacids. Other polymeric compositions having comparable properties mayalso find use. The polyamide compositions may be modified to introduceother functional groups which provide for charged structures, so thatthe surfaces of the matrix may be neutral, positive or negative, as wellas neutral, basic or acidic. Preferred surfaces are positively charged.It has been determined that this positive charge enhances both stabilityand shelf-life.

When used with whole blood, the porous matrix preferably has pores withan average diameter in the range of from about 0.1 to 2.0 μm, morepreferably from about 0.6 to 1.0 μm. When the porous matrix containspores having an average diameter of about 0.8 μm, the sample of bloodwill not cause a chromatographic effect. That is, the blood sample willnot seek out the edges of the circular matrix. Rather, the blood remainsseated within all the pores of the matrix and provides for a uniformreadability of the entire matrix. In addition, this pore size maximizesthe non-blotting effect of the blood. That is, the pore size is bothadequately filled, but not overfilled, so that the hematocrit level ofblood will not cause the sample to require blotting prior to reading ofthe sample. Also, it has been found that pores of this size are optimalwhen shelf-life and stability are taken into consideration.

A preferred manner of preparing the porous material is to cast thehydrophilic polymer onto a core of non-woven fibers. The core fibers canbe any fibrous material that produce the described integrity andstrength, such as polyesters and polyamides. The reagent that will formthe light-absorbing reaction product, which is discussed later indetail, is present within the pores of the matrix but does not block thematrix so that the liquid portion of the assay medium, e.g. blood, beinganalyzed can flow through the pores of the matrix, while particles, suchas erythrocytes, are held at the surface.

The matrix is substantially reflective so that it gives a diffusereflectance without the use of a reflective backing. Preferably at least25%, more preferably at least 50%, of the incident light applied to thematrix is reflected and emitted as diffuse reflectance. A matrix of lessthan about 0.5 mm thickness is usually employed, with from about 0.01 mmto about 0.3 mm being preferred. A thickness of from about 0.1 mm toabout 0.2 mm is most preferred, particularly for a nylon matrix.

Typically, the matrix will be attached to a holder in order to give itphysical form and rigidity, although this may not be necessary. FIG. 1shows one embodiment of the invention in which there is a strip 10having a thin hydrophilic matrix pad 11 is positioned at one end of aplastic holder or handle 12 by means of an adhesive 13 which directlyand firmly attaches the reagent pad 11 to the handle 12. A hole 14 ispresent in the plastic holder 12 in the area to which reagent pad 11 isattached so that sample can be applied to one side of the reagent padand light reflected from the other side.

A liquid sample to be tested is applied to pad 11.

Generally, with blood being exemplary of a sample being tested, thereagent pad will be on the order of about 10 mm² to 100 mm² in surfacearea, especially 10 mm² to 50 mm² in area (or having a diameter of about2 mm to about 10 mm), which is normally a volume that 5-10 microlitersof sample will more than saturate. Of course, once saturation is reachedat above the threshold of about 5-10 microliters, no other requirementof blood amount is necessary.

Diffuse reflectance measurements in the prior art have typically beentaken using a reflective backing attached to or placed behind thematrix. No such backing is needed or will normally be present during thepractice of the present invention, either as part of the reagent elementor the reflectance apparatus.

As can be seen from FIG. 1, the support holds reagent pad 11 so that asample can be applied to one side of the reagent pad 11 while lightreflectance is measured from the side of the reagent pad 11 opposite thelocation where sample is applied.

FIG. 2 shows a system in which the reagent is applied to the side withthe hole 14 in the backing handle 12 while light is reflected andmeasured on the other side of the reagent pad 11. Other structures thanthe one depicted may be employed. The pad 11 may take various shapes andforms, subject to the limitations provided herein. The pad 11 will beaccessible on at least one surface and usually two surfaces.

The hydrophilic layer (reagent element) may be attached to the supportby any convenient means, e.g., a holder, clamp or adhesives; however, inthe preferred method it is bonded to the backing. The bonding can bedone with any non-reactive adhesive, by a thermal method in which thebacking surface is melted enough to entrap some of the material used forthe hydrophilic layer, or by microwave or ultrasonic bonding methodswhich likewise fuse the hydrophilic sample pads to the backing. It isimportant that the bonding be such as to not itself interferesubstantially with the diffuse reflectance measurements or the reactionbeing measured, although this is unlikely to occur as no adhesive needbe present at the location where the reading is taken. For example, anadhesive 13 can be applied to the backing strip 12 followed first bypunching hole 14 into the combined strip and adhesive and then applyingreagent pad 11 to the adhesive in the vicinity of hole 14 so that theperipheral portion of the reagent pad attaches to the backing strip.

The Chemical Reagents

Any signal producing system may be employed that is capable of reactingwith the analyte in the sample to produce (either directly orindirectly) a compound that is characteristically absorptive at awavelength other than a wavelength at which the assay mediumsubstantially absorbs.

Polyamide matrices are particularly useful for carrying out reactions inwhich a substrate (analyte) reacts with an oxygen-utilizing oxidaseenzyme in such a manner that a product is produced that further reactswith a dye intermediate to either directly or indirectly form a dyewhich absorbs in a predetermined wavelength range. For example, anoxidase enzyme can oxidize a substrate and produce hydrogen peroxide asa reaction product. The hydrogen peroxide can then react with a dyeintermediate or precursor, in a catalysed or uncatalyzed reaction, toproduce an oxidized form of the intermediate or precursor. This oxidizedmaterial may produce the colored product or react with a secondprecursor to form the final dye.

Nonlimiting examples of analyses and typical reagents include thefollowing materials shown in the following list:

Analyte and Sample Type Reagents Glucose in blood, serum, GlucoseOxidase, Peroxidase urine or other biological and an Oxygen Acceptorfluids, wine, fruit juices Oxygen Acceptors include: or other coloredaqueous O-dianisidine (1) fluids. Whole blood is a O-toluidineparticularly preferred O-tolidine (1) sample type, as separationBenzidine (1) is time-consuming and 2,2′-Azinodi-(3-ethylbenz-impractical with home use. thiazoline sulphonic acid-(6)) (1)3-Methyl-2-benzothiazolinone hydrazone plus N,N-dimethylaniline (1)Phenyl plus 4-aminophenazone (1) Sulfonated 2,4-dichlorophenol plus4-aminophenazone (2) 3-Methyl-2-benzothiazolinone hydrazone plus3-(dimethylamino)benzoic acid (3) 2-Methoxy-4-allyl phenol (4)4-Aminoantipyrene-dimethylaniline (5) (1) As reported ClinicalChemistry, Richterich and Columbo, p. 367 and references cited therein.(2) Analyst, 97, (1972) 142-5. (3) Anal. Biochem., 105, (1980) 389-397.(4) Anal. Biochem., 79, (1977) 597-601. (5) Clinica Chemica Acta, 75,(1977) 387-391 All incorporated herein by reference.

The Analysis Method

The analysis method of this invention relies on a change in absorbance,as measured by diffuse reflectance, which is dependent upon the amountof analyte present in a sample being tested. This change may bedetermined by measuring the change in the absorbance of the test samplebetween two or more points in time.

The first step of the assay to be considered will be application of thesample to the matrix. In practice, an analysis could be carried out asfollows: First a sample of aqueous fluid containing an analyte isobtained. Blood may be obtained by a finger stick, for example. Anexcess over threshold matrix saturation in the area where reflectancewill be measured (i.e., about 5-10 microliters) of this fluid is appliedto the regent element or elements of the test device. Simultaneousstarting of a timer is not required (as is commonly required in theprior art), as will become clear below, due to the initializationprocedure practiced by the present invention. Excess fluid can beremoved, such as by light blotting, but such removal is also notrequired. The test device is typically mounted in an instrument forreading light absorbance, e.g., color intensity by reflectance, prior toapplication of the sample. Absorbance is measured at certain points intime after application of the sample. Absorbance refers in thisapplication not only to light within the visual wavelength range butalso outside the visual wavelength range, such as infrared andultraviolet radiation. From these measurements of absorbance a rate ofcolor development can be calibrated in terms of analyte level.

The Measuring Instrument

A suitable instrument, such as a diffuse reflectance spectrophotometerwith appropriate software, can be made to automatically read reflectanceat certain points in time, calculate rate of reflectance change, and,using calibration factors, output the level of analyte in the aqueousfluid. Such a device is schematically shown in FIG. 2 wherein a testdevice of the invention comprising backing 12 to which reagent pad 11 isaffixed is shown. Light source 5, for example a high intensity lightemitting diode (LED) projects a beam of light onto the reagent pad. Asubstantial portion (at least 25%, preferably at least 35%, and morepreferably at least 50%, in the absence of reaction product) of thislight is diffusively reflected from the reagent pad and is detected bylight detector 6, for example a phototransistor that produces an outputcurrent proportional to the light it receives.

Light source 5 and/or detector 6 can be adapted to generate or respondto a particular wavelength light, if desired. The output of detector 6is passed to amplifier 7, for example, a linear integrated circuit whichconverts the phototransistor current to a voltage. The output ofamplifier 7 can be fed to track and hold circuit 8. This is acombination linear/digital integrated circuit which tracks or followsthe analog voltage from amplifier 7 and, upon command frommicroprocessor 20, locks or holds the voltage at its level at that time.

Analog-to-digital converter 19 takes the analog voltage from track andhold circuit 8 and converts it to, for example, a twelve-bit binarydigital number upon command of microprocessor 20. Microprocessor 20 canbe a digital integrated circuit. It serves the following controlfunctions: 1) timing for the entire system; 2) reading of the output ofanalog/digital converter 19; 3) together with program and data memory21, storing data corresponding to the reflectance measured at specifiedtime intervals; 4) calculating analyte levels from the storedreflectances; and 5) outputting analyte concentration data to display22. Memory 21 can be a digital integrated circuit which stores data andthe microprocessor operating program. Reporting device 22 can takevarious hard copy and soft copy forms. Usually it is a visual display,such as a liquid crystal (LCD) or LED display, but it can also be a tapeprinter, audible signal, or the like. The instrument also can include astart-stop switch and can provide an audible or visible time output toindicate times for applying samples, taking readings etc., if desired.

Reflectance Switching

In the present invention, the reflectance circuit itself can be used toinitiate timing by measuring a drop in reflectance that occurs when theaqueous portion of the suspension solution applied to the reagent pad(e.g., blood) migrates to the surface at which reflectance is beingmeasured. Typically, the measuring device is turned on in a “ready” modein which reflectance readings are automatically made at closely spacedintervals (typically about 0.2 seconds) from the typically off-white,substantially dry, unreacted reagent strip. The initial measurement istypically made prior to penetration of the matrix by fluid beinganalyzed but can be made after the fluid has been applied to a locationon the reagent element other than where reflectance is being measured.The reflectance value is evaluated by the microprocessor, typically bystoring successive values in memory and then comparing each value withthe initial unreacted value. When the aqueous solution penetrates thereagent matrix, the drop in reflectance signals the start of themeasuring time interval. Drops in reflectance of 5-50% can be used toinitiate timing, typically a drop of about 10%. In this simple way thereis exact synchronization of assay medium reaching the surface from whichmeasurements are taken and initiation of the sequence of readings, withno requirement of activity by the user.

Although the total systems described in this application areparticularly directed to the use of polyamide matrices and particularlyto the use of such matrices in determining the concentration of varioussugars, such as glucose, and other materials of biological origin, thereis no need to limit the reflectance switching aspect of the invention tosuch matrices. For example, the matrix used with reflectance switchingmay be formed from any water-insoluble hydrophilic material and anyother type of reflectance assay.

Particular Application to Glucose Assay

A particular example with regard to detecting glucose in the presence ofred blood cells will now be given in order that greater detail andparticular advantage can be pointed out. Although this represents apreferred embodiment of the present invention, the invention is notlimited to the detection of glucose in blood.

The use of polyamide surfaces to form the reagent element provides anumber of desirable characteristics in the present invention. These arethat the reagent element is hydrophilic (i.e., takes up reagent andsample readily), does not deform on wetting (so as to provide a flatsurface for reflectance reading), is compatible with enzymes (in orderto impart good shelf stability), takes up a limited sample volume perunit volume of membrane (necessary in order to demonstrate an extendeddynamic range of measurements), and shows sufficient wet strength toallow for routine manufacture.

In a typical configuration, the method is carrier out using an apparatusconsisting of a plastic holder and the reagent element (the matrixhaving the signal producing system impregnated therein.) The preferredmatrix for use in preparing the reagent element is a nylonmicrofiltration membrane, particularly membranes made from nylon-66 caston a core of non-woven polyester fibers. Numerous nylon microfiltrationmembranes of this class are produced commercially by the Pall UltrafineFiltration Corporation, having average pore sizes from 0.1 to 3.0microns. These materials shown mechanical strength and flexibility,dimensional stability upon exposure to water, and rapid wetting.

Many variations in specific chemical structure of the nylon arepossible. These include unfunctionalized nylon-66 with charged endgroups (sold under the trademark ULTRAPORE by Pall Ultrafine FiltrationCorporation, “Pall”). Positive charges predominate below pH 6 whilenegative charges predominate above pH 6. In other membranes the nylon isfunctionalized before the membrane is formed to give membranes withdifferent properties. Nylons functionalized with carboxy groups arenegatively charged over a wide pH range (sold as CARBOXYDYNE by Pall).Nylons can also be functionalized with a high density of positivelycharged groups on its surface, typically quaternary amine groups, sothat they display little variation in charge over a wide pH range (soldas POSIDYNE by Pall). Such materials are particularly well suited forthe practice of the present invention.

It has been found that keeping the pH of the solution below 4.8 willhelp stabilize the enzymes in solution. The most efficient level ofstability has been found at pH 4.0. This results in shelf life at roomtemperature of 12-18 months. Consequently, a strip with positivelycharged ions is most desirable.

It is also possible to use membranes having reactive functional groupsdesigned for covalent immobilization of proteins (sold as BIODYNE IMMUNOAFFINITY membranes by Pall). Such materials can be used to covalentlyattach proteins, e.g. enzymes, used as reagents. Although all of thesematerials are usable, nylon having a high density of positively chargedgroups on its surface provide the best stability of reagents whenformulated into a dry reagent pad. Unfunctionalized nylon gives the nextbest stability with the carboxylated nylons next best.

Desirable results can be obtained with pore sizes ranging from about0.2-2.0 μm, preferably about 0.5-1.2 μm, and most preferably about 0.8μm, when used with whole blood.

The form of the handle on which the reagent element is assembled isrelatively unimportant as long as the handle allows access to one sideof the reagent element by sample and to the other side of the reagentelement by incident light whose reflectance is being measured. Thehandle also aids in inserting the reagent element into the absorbancemeasuring device so that it registers with the optical system. Oneexample of a suitable handle is a mylar or other plastic strip to whicha transfer adhesive such as 3M 465 or Y9460 transfer adhesive has beenapplied. A hole is punched into the plastic through the transferadhesive. A reagent element, typically in the form of a thin pad, eithercontaining reagents or to which reagents will later be added, is thenapplied to the handle by means of the transfer adhesive so that it isfirmly attached to the handle in the area surrounding the hole that hasbeen punched through the handle and the transfer adhesive.

Such a device is illustrated in FIG. 1, which shows a strip 10 having areagent pad 11 attached to a Mylar handle 12 by means of adhesive 13.Hole 14 allows access of the sample or incident light to one side ofreagent pad 11 while access to the other side of the reagent pad isunrestricted. All dimensions of the reagent pad and handle can beselected so that the reagent pad fits securely into areflectance-reading instrument in proximal location to a light sourceand a reflected-light detector. Generally, dimensions of the hole are inthe range of about 2-10 mm diameter, and that of the width of the handleabout 15 mm. A 5 mm diameter hole 14 in the reagent strip shown in FIG.1 works quite satisfactorily. Naturally, there is no particular limit onthe minimum diameter of such a hole, although diameters of at least 2 mmare preferred for ease of manufacture, sample application, and lightreflectance reading.

As further seen in FIGS. 3 and 4, the strip 10 can be optimally guidedinto a slot 50 on scanning machine 60. This is accomplished by placing anotch 15 in the strip 10 at about the midpoint of the top of strip 10.In so doing, the strip 10, when guided through sides 55 of slot 50, willarrive repeatably at the same location, to assure high assurance in testresults. Such repeatability is accomplished by moving the notch 15against post 65. The strip 10 will pivot around the post 65 at the notch15, so that the edges 16 of the strip will fit within the sides 55 ofthe slot 50. This, of course, also repeatably aligns the hole 14 overthe test center 80 comprising multiple LEDs 5 in the scanning machine60. This insures that the hole 14 containing a blood sample will haveuniform dosage of incident light for analysis.

Although a number of dyes could be used as indicators, the choice willdepend upon the nature of the sample. It is necessary to select a dyehaving an absorbance at a wavelength different from the wavelength atwhich red blood cells absorb light, with whole blood as the assay mediumor other contaminants in the solution being analyzed with other assaymedia. The MBTH-DMAB dye couple (3-methyl-2-benzothiazolinone hydrazonehydrochloride and 3-dimethylaminobenzoic acid), although beingpreviously described as suitable for color development for peroxidaselabels in enzyme immunoassays, has never been used in a commercialglucose measuring reagent. This dye couple gives greater dynamic rangeand shows improved enzymatic stability as compared to traditional dyesused for glucose measurement, such as benzidine derivatives. Thisenzymatic stability also makes the MBTH-DMAB dye couple especiallydesirable in order to insure longer shelf life of the test strips.Furthermore, the MBTH-DMAB dye couple is not carcinogenic, acharacteristic of most benzidine derivatives.

Another dye couple that can be used in the measurement of glucose is theAAP-CTA (4-aminoantipyrene and chromotropic acid) couple. Although thiscouple does not provide as broad a dynamic range as MBTH-DMAB, it isstable and suitable for use in the practice of the present inventionwhen measuring glucose. Again, the AAP-CTA dye couple provides anexpanded dynamic range and greater enzymatic activity stability than themore widely used benzidine dyes.

The use of the MBTH-DMAB couple allows for correction of hematocrit anddegree of oxygenation of blood with a single correction factor. The moretypically used benzidine dyes do not permit such a correction. TheMBTH-DMAB dye forms a chromophore that absorbs at approximately 635 nmbut not to any significant extent at 700 nm. Slight variations inmeasuring wavelengths (± about 10 nm) are permitted. At 700 nm bothhematocrit and degree of oxygenation can be measured by measuring bloodcolor. Furthermore, light emitting diodes (LED) are commerciallyavailable for both 635 nm and 700 nm measurements, thereby simplifyingmass-production of a device. By using the preferred membrane pore sizedescribed above and the subject reagent formulation, both hematocrit andoxygenation behavior can be corrected by measuring at the single 700 nmwavelength.

Two additional conditions were found to provide particular stability andlong shelf life for a glucose oxidase/peroxidase formulation on apolyamide matrix. Storage is enhanced at a pH in the range of 3.8 to 5.0preferably about 3.8 to 4.3, most preferably about 4.0. Similarly,unexpectedly good storage and stability was found with mixture of aconcentrated buffer system to the the reagents found in the matrix. Themost effective buffer was found to be a 10% weight citrate buffer, withconcentrations from about 5-15% being effective. These are weight/volumepercentages of the solution in which the reagents are applied to thematrix. Other buffers can be used on the same molar basis. Greateststability was achieved using a low pH, preferably about pH 4, anMBTH-DMAB dye system, and a high enzyme concentration of approximately500-1000 M/ml of application solution. As previously indicated, suchstrips prepared using these parameters result in shelf life of about12-18 months.

In preparing the MBTH-DMAB reagent and the enzyme system that forms theremainder of the signal producing system, it is not necessary tomaintain exact volumes and ratios although the suggested values belowgive good results. Reagents are readily absorbed by the matrix pad whenthe glucose oxidase is present in a solution at about 27-54% by volume,the peroxidase is present at a concentration of about 2.7-5.4 mg/ml,MBTH is present at a concentration of about 4-8 mg/ml, and DMAB ispresent at a concentration of about 8-16 mg/ml. The DMAB-MBTH weightratio is preferably maintained in the range of 1:1 to 4:1, preferablyabout 1.5:1 to 2.5:1, most preferably about 2:1.

The basic manufacturing techniques for the reagent element are, onceestablished, straightforward. The membrane itself is strong and stable,particularly when a nylon membrane of the preferred embodiment isselected. Only two solutions are necessary for applying reagent, andthese solutions are both readily formulated and stable. The firstgenerally contains the dye components and the second generally containsthe enzymes. When using the MBTH-DMAB dye couple, for example, theindividual dyes are dissolved in an aqueous organic solvent, typically a1:1 mixture of acetonitrile and water. The matrix is dipped into thesolution, excess liquid is removed by blotting, and the matrix is thendried, typically at 50° C.-60° C. for 10-20 minutes. The matrixcontaining the dyes is then dipped into an aqueous solution containingthe enzymes. A typical formulation would contain the peroxidase andglucose oxidase enzymes as well as any desired buffer, preservative,stabilizer, or the like. The matrix is then blotted to remove excessliquid and dried as before. A typical formulation for the glucosereagent is as follows:

Dye Dip

Combine:

-   -   40 mg MBTH,    -   80 mg DMAB,    -   5 ml acetonitrile, and    -   5 ml water.        Stir until all solids are dissolved and pour onto a glass plate        or other flat surface. Dip a piece of Posidyne membrane (Pall        Co.), blot off excess liquid, and dry at 56° C. for 15 minutes.

Enzyme Dip

Combine:

-   -   6 ml water,    -   10 mg EDTA, disodium salt,    -   200 mg Sigma Poly Pep™, low viscosity,    -   0.668 g sodium citrate,    -   0.523 g citric acid,    -   2.0 ml 6 wt % GAF Gantrez™ AN-139 dissolved in water    -   30 mg horseradish peroxidase, 100 units/mg, and    -   3.0 ml glucose oxidase, 2000 units/ml.        Stir until all solids are dissolved and pour onto a glass plate        or other flat surface. Dip a piece of membrane previously        impregnated with dyes, blot off excess liquid, and dry at 56° C.        for 15 minutes.

The electronic apparatus used to make the reflectance readings minimallycontains a light source, a reflected light detector, an amplifier, ananalog to digital converter, a microprocessor with memory and program,and a display device, as seen in FIG. 2.

The light source typically consists of a light emitting diode (LED).Although it is possible to use a polychromatic light source and a lightdetector capable of measuring at two different wavelengths, a preferredapparatus would contain two LED sources or a single diode capable ofemitting two distinct wavelengths of light. Commercially available LEDsproducing the wavelengths of light described as being preferred in thepresent specification include a Hewlett Packard HLMP-1340 with anemission maximum at 635 nm and a Hewlett Packard QEMT-1045 with anarrow-band emission maximum at 700 nm. Suitable commercially availablelight detectors include a Hammamatsu 5874-18K and a Litronix BPX-65.

Although other methods of taking measurements are feasible, thefollowing method has provided the desired results. Readings are taken bythe photodetector at specified intervals after timing is initiated. The635 nm LED is powered only during a brief measuring time span thatbegins approximately 20 seconds after the start time as indicated by thepreviously described reflectance switching system. If this readingindicates that a high level of glucose is present in the sample, a30-second reading is taken and used in the final calculation in order toimprove accuracy. Typically, high levels are considered to begin atabout 250 mg/dl. The background is corrected with a 700 nm reading takenabout 15 seconds after the start of the measurement period. The readingfrom the photodetector is integrated over the interval while theappropriate LED is activated, which is typically less than one second.The raw reflectance readings are then used for calculations performed bythe microprocessor after the signal has been amplified and converted toa digital signal. Numerous microprocessors can be used to carry out thecalculation. An AIM65 single-board microcomputer manufactured byRockwell International has proven to be satisfactory.

The present methods and apparatuses allow a very simple procedure withminimum operational steps on the part of the user. In use, the reagentstrip 10 is placed in the detector so that the hole 14 in the strip 10registers with the optics of the detecting system. The above-describednotch 15/post 65 system, as seen in FIGS. 4 and 5 works nicely toaccomplish such alignment. A removable cap or other cover 90 is placedover the optics and strip to shield the assembly from ambient light.This is done to enhance reading of the strip 10. While the initilizationprocess can begin in light, direct sunlight or high intensity room lighttends to inhibit results. The cap 90 insures that direct light does nothit the reagent strip 10. The cap 90 need not be light-tight, onlyenough to protect the strip 10 from direct light.

The measurement sequence is then initiated by pressing a button on themeasuring apparatus that activates the microcomputer to take ameasurement of reflected light from the unreacted reagent pad, called anR_(dry) reading. The cap 90 is then removed and a drop of blood isapplied to the reagent strip 10, typically while the reagent strip 10 isregistered with the optics and the reading device. It is preferred thatthe reagent strip be left in register with the optics in order tominimize handling. The cap 90 is then closed.

The instrument is capable of sensing the application of blood or othersample by a decrease in the reflectance when the sample passes throughthe matrix and reflected light is measured on the opposite side. Thedecrease in reflectance initiates a timing sequence which is describedin detail at other locations in this specification. The cap 90 should bereplaced within 15 seconds of sample application, although this time mayvary depending on the type of sample being measured.

Results are typically displayed at approximately 30 seconds after bloodapplication when a blood glucose sample is being measured, although a 20second reaction is permissible for glucose samples having aconcentration of glucose of less than 250 mg/dl. If other samples arebeing measured, suitable times for displaying the result may differ andcan be readily determined from the characteristics of the reagent/sampleselected.

A particularly accurate evaluation of glucose level (or any otheranalyte being measured) can be made using the background current, i.e.,the current from the photo detector with power on but with no lightreflected from the reagent pad, in order to make a backgroundcorrection. It has been demonstrated that over a 2-3 month period thatthis value does not change for a particular instrument preparedaccording to the preferred embodiments of this specification, and it ispossible to program this background reading into the computer memory asa constant.

With a slight modification of the procedure, however, this value can bemeasured (or normalized) with each analysis for more accurate results.Each LED is turned on prior to placement of the blood sample on thereagent strip 10 but with the reagent strip 10 in place. A reflectancevalue of the strip 10 is then measured, with the reagent strip 10 inplace and the light protective cap 90 closed. If this measurement isdifferent than the original measurement of the reflectance value, powerto the LED is increased so that the reflectance will be the same. Thisinsures that the measurement of blood glucose content is being made onthe same repeatable scale for each blood glucose reading.

The reason for instituting this method is twofold. First, the intensityof light emitting diodes will vary greatly from LED to LED, even whenall the measuring LEDs are new. Second, the LED efficiency will varywith both temperature and the life of the LED. With this method, resultsare repeatable on the same scale.

The raw data necessary for calculating a result in a glucose assay are abackground current reported as background reflectance, R_(b), asdescribed above; a reading of the unreacted test strip, R_(dry), whichis about 95% opaque to light and is also described above; and an endpoint measurement. Using the preferred embodiments described herein, theend point is not particularly stable and must be precisely timed fromthe initial application of blood. However, the meter as described hereinperforms this timing automatically. For glucose concentrations below 250mg/dl, a suitably stable end point is reached in 20 seconds, and a finalreflectance, R₂₀, is taken. For glucose concentrations up to 450 mg/dl,a 30-second reflectance reading, R₃₀, is adequate. Although the systemdescribed herein displays good differentiation up to 800 mg/dl ofglucose, the measurement is somewhat noisy and inaccurate above 450mg/dl, although not so great as to cause a significant problem. Longerreaction times should provide more suitable readings for the higherlevels of glucose concentration.

The 700 nm reflectance reading for the dual wavelength measurement istypically taken at 15 seconds (R₁₅). By this time blood will havecompletely saturated the reagent pad. Beyond 15 seconds the dye reactioncontinues to take place and is sensed, to a small part, by a 700 nmreading. Accordingly, since dye absorption by the 700 nm signal is adisadvantage, readings beyond 15 seconds are ignored in thecalculations.

The raw data described above are used to calculate parametersproportional to glucose concentration which can be more easilyvisualized than reflectance measurements. A logarithmic transformationof reflectance analogous to the relationship between absorbance andanalyte concentration observed in transmission spectroscopy (Beer's Law)can be used if desired. A simplification of the Kubelka-Monk equations,derived specifically for reflectance spectroscopy, have provenparticularly useful. In this derivation K/S is related to analyteconcentration with K/S defined by Equation 1.K/S−t=(1−R*t)²/(2×R*t)  (1)R*t is the reflectivity taken at a particular end point time, t, and isthe absorbed fraction of the incident light beam described by Equation2, where R_(t) is the end point reflectance, R₂₀ or R₃₀.R*t=(R _(t) −R _(b))/(R _(dry) −R _(b))  (2)R*t varies from 0 for no reflected light (R_(b)) to 1 for totalreflected light (R_(dry)). The use of reflectivity in the calculationsgreatly simplifies meter design as a highly stable source and adetection circuit become unnecessary since these components aremonitored with each R_(dry) and R_(b) measurement.

For a single wavelength reading K/S can be calculated at 20 seconds(K/S-20) or 30 seconds (K/S-30). The calibration curves relating theseparameters to YSI (Yellow Springs Instruments) glucose measurements canbe precisely described by the third order polynomial equation outlinedin Equation 3.YSI=a ₀ +a ₁(K/S)+a ₂(K/S)² +a ₃(K/S)³  (3)

The coefficients for these polynomials are listed in Table 1.

TABLE 1 Coefficients for Third Order Polynomial Fit of Single WavelengthCalibration Curves K/S-20 K/S-30 a₀ −55.75 −55.25 a₁ 0.1632 0.1334 a₂−5.765 × 10⁻⁵    −2.241 × 10⁻⁵    a₃ 2.58 × 10⁻⁸ 1.20 × 10⁻⁸

The single chemical species being measured in the preferred embodimentsis the MBTH-DMAB inamine dye and the complex matrix being analyzed iswhole blood distributed on a 0.8μ Posidyne™ membrane. A review entitled“Application of Near Infra Red Spectrophotometry to the NondestructiveAnalysis of Foods: A Review of Experimental Results”, CRC CriticalReviews in Food Science and Nutrition, 18(3) 203-30 (1983), describesthe use of instruments based on the measurement of an optical densitydifference ΔOD (λ_(a)−λ_(b)) where ODλ_(a) is the optical density of thewavelength corresponding to the absorption maximum of a component to bedetermined and ODλ_(b) is the optical density at a wavelength where thesame component does not absorb significantly.

The algorithm for dual wavelength measurement is by necessity morecomplex than for single wavelength measurement but is much morepowerful. The first order correction applied by the 700 nm reading is asimple subtraction of background color due to blood. In order to makethis correction, a relationship between absorbance at 635 nm and 700 nmdue to blood color can be and was determined by measuring blood sampleswith 0 mg/dl glucose over a wide range of blood color. The color rangewas constructed by varying hematocrit, and fairly linear relationshipswere observed. From these lines the K/S-15 at 700 nm was normalized togive equivalence to the K/S-30 at 635 nm. This relationship is reportedin Equation 4, where K/S-15n is the normalized K/S-15 at 700 nm.K/S-15n=(K/S-15×1.54)−0.133  (4)

Note that the equivalence of the normalized 700 nm signal and the 635 nmsignal is only true at zero glucose. The expressions from which thecalibration curves were derived are defined by Equations 5 and 6.K/S-20/15=(K/S-20)−(K/S-15n)  (5)K/S-30/15=(K/S-30)−(K/S-15n)  (6)

These curves are best fit by fourth-order polynomial equations similarto Equation 3 to which a fourth-order term in K/S is added. Thecomputer-fit coefficients for these equations are listed in Table 2.

TABLE 2 Coefficients for Fourth-Order Polynomial Fit of Dual WavelengthCalibration Curves K/S-20/15 K/S-30/15 a₀ −0.1388 1.099 a₁ 0.10640.05235 a₂ 6.259 × 10⁻⁵ 1.229 × 10⁻⁴ a₃ −6.12 × 10⁻⁸ −5.83 × 10⁻⁸ a₄   3.21 × 10⁻¹¹   1.30 × 10⁻¹¹

A second order correction to eliminate errors due to chromatographyeffects has also been developed. Low hematocrit samples havecharacteristically low 700 nm readings compared to higher hematocritsamples with the same 635 nm reading. When the ratio of(K/S-30)/(K/S-15) is plotted versus K/S-30 over a wide range ofhematocrits and glucose concentrations, the resulting line on the graphindicates the border between samples which display chromatographyeffects (above the curve) and those that do not (below the curve). TheK/S-30 for the samples above the curve are corrected by elevating thereading to correspond to a point on the curve with the same(K/S-30)/(K/S-15), as demonstrated by the correction made in FIG. 5.

The correction factors reported above were tailored to fit a singleinstrument and a limited number of reagent preparations. The algorithmcan be optimized for an individual instrument and reagent in the samemanner that is described above.

In summary, the system of the present invention minimizes operatoractions and provides numerous advantages over prior artreflectance-reading methods. When compared to prior methods fordetermining glucose in blood, for example, there are several apparentadvantages. First, the amount of sample required to saturate the thinreagent pad is small (typically 5-10 microliters), and is of course,volume independent once the threshold volume of blood is supplied to thereagent pad. Second, operator time required is only that necessary toapply the sample to the thin hydrophilic layer and close the cover(typically 4-7 seconds). Third, no simultaneous timing start isrequired. Fourth, whole blood can be used. The method does not requireany separation or utilization of red-cell-free samples and likewise canbe used with other deeply colored samples. Fifth, via the reflectancereading and normalization techniques applied in the present inventionthe system provides reliable, accurate, repeatable readings for thelifetime of the scanning system.

Several unobvious advantages arise as a result of the practice of thepresent invention with whole blood. Normally, aqueous solutions (likeblood) will penetrate a hydrophilic membrane to give a liquid layer onthe opposite side of the membrane, a surface that is then not suited fora reflectance measurement. It has been discovered, however, that blood,apparently because of interactions of red blood cells and proteins inthe blood with the matrix, will wet the polyamide matrix without havingan excess liquid penetrate the porous matrix to interfere with thereflectance reading on the opposite side of the matrix.

Furthermore, the thin membranes used in the present invention would beexpected when wet to transmit light and return only a weak signal to thereflectance measuring device. Prior teachings have generally indicatedthat a reflective layer is necessary behind the matrix in order toreflect sufficient light. In other cases a white pad has been placedbehind the reagent pad prior to color measurement. In the present case,neither a reflective layer nor a white pad is required. In fact, theinvention is typically carried out with a light-absorbing surface behindthe reagent element when incident light is impinged upon the matrix.This is accomplished using a light absorbing surface behind the reagentelement, coupled with measuring reflectance at two differentwavelengths. It allows acceptable reflectance measurements to beobtained without removal of excess liquid from the matrix, therebyeliminating a step typically required by previous teachings.

The invention now being generally described, the same will be betterunderstood by reference to the following specific examples which arepresented for purposes of illustration only and are not to be consideredlimiting of the invention unless so specified.

EXAMPLE I

Reproducibility

One male blood sample (having a hematocrit level of 45) was used tocollect the reproducibility data using the presently preferredembodiment of the system, called the MPX system. The results are setforth in Tables 3-5.

TABLE 3 Reproducibility of a Single Wavelength System Average (mg/dl)*S.D. (mg/dl) % C.V.** ***YSI(mg/dl) 20 sec. 30 sec. 20 sec. 30 sec. 20sec. 30 sec. 25 23.1 23.0 2.1 2.04 9.1 9.0 55 53.3 53.2 3.19 3.32 6.06.3 101 101 101 3.0 3.3 3.0 3.0 326 326.6 327 13.3 9.8 4.1 3.0 501 50317.1 3.4 690 675 28 4.15 810 813 37 4.5 *S.D. = Standard Deviation**%C.V. = Covariance (measured by percentage) ***YSI = Yellow SpringInstrument Glucose reading

TABLE 4 Reproducibility of Dual Wavelength System Average (mg/dl) S.D.(mg/dl) % C.V. YSI(mg/dl) 20 sec. 30 sec. 20 sec. 30 sec. 20 sec. 30sec. 25 25 27 1.34 1.55 5.4 5.7 55 55 57.4 2.58 2.62 4.7 4.6 101 101101.5 2.55 2.18 2.5 2.1 326 332 330 15.0 7.1 4.5 2.1 501 505 21.3 4.2690 687 22.8 3.3 810 817 30.4 3.7

TABLE 5 Reproducibility of a 3.0 mm Diameter Aperature % C.V. YSI(mg/dl) 4.7 mm 3.0 mm 55-100 4.8 4.9 300 3.0 5.0 600 3.8 5.5 avg. 3.95.1

The blood was divided into aliquots and spiked with glucose across arange of 25-800 mg/dl. Twenty determinations were made at each glucosetest level from strips taken at random from a 500 strip sample (LotFJ4-49B). The results of this study lead to the following conclusions:

-   -   1. Single vs. Dual Wavelength: The average covariance for the        30-second dual result was 3.7% vs. 4.8% for the 30-second single        wavelength result, an improvement of 23% across a glucose range        of 25-810 mg/dl. There was a 33% improvement in covariance in        the 25-326 mg/dl glucose range. Here the covariance decreased        from 5.4% to 3.6%, a significant improvement in the most used        range. The 20-second dual wavelength measurement gave similar        improvements in covariance compared to the single wavelength        measurement in the 25-326 mg/dl range (Tables 3 and 4).    -   2. Dual Wavelength, 20 vs. 30-second Result: The average        covariance for a 20-second result in the 25-100 mg/dl range is        nearly identical to the 30-second reading, 4.2% vs. 4.1%.        However, at 326 mg/dl the 30-second reading has a covariance of        2.1% and the 20-second result a covariance of 4.5%. As was seen        in the K/S-20 response curve, the slope begins to decrease        sharply above 250 mg/dl. This lead to poor reproducibility at        glucose levels greater than 300 for the 20-second result. From        this reproducibility data the cutoff for the 20-second result is        somewhere between 100 and 326 mg/dl. A cutoff of 250 mg/dl was        later determined from the results of the recovery study set        forth in Example II.    -   3. Aperture Size: A smaller optics aperture size, 3.0 mm, was        investigated. Initial experimentation using a 10 replicate,        hand-dipped disk sample did show improved covariances with the        3.0 mm aperture, apparently because of easier registration with        the system optics. However, when machine-made roll membrane was        used, the average (Table 5) of the larger aperture, 4.7 mm, was        3.9% vs. an average covariance for the 3.0 mm aperture of 5.1%.        This 30% increase was probably due to the uneven surface of the        roll membrane lot as discussed below.

EXAMPLE II

Recovery

For comparison of the present preferred method called MPX against atypical prior art method using a Yellow Springs Instrument Model 23Aglucose analyzer manufactured by Yellow Springs Instrument Co., YellowSprings, Ohio (YSI), blood from 36 donors was tested. The donors weredivided equally between males and females and ranged in hematocrit from35 to 55%. The blood samples were used within 30 hours of collection,with lithium heparin as the anti-coagulant. Each blood sample wasdivided into aliquots and spiked with glucose to give 152 samples in therange of 0-700 mg/dl glucose. Each sample was tested in duplicate for atotal of 304 data points.

Response curves were constructed for the appropriate equation (seeTables 1 and 2). These MPX glucose values were then plotted vs. the YSIvalues to give scattergrams, as seen in FIGS. 6 a and 6 b for the SingleWavelength System, and FIGS. 7 a through 7 d for the Dual WavelengthSystem.

Comparison of MPX Systems: For both the 20-second and 30-secondmeasurement times there is visually more scatter in thesingle-wavelength scattergrams than the dual-wavelength scattergrams.The 20-second reading becomes very scattered above 250 mg/dl but the30second measurement does not have wide scatter until the glucose levelis greater than 500 mg/dl.

These scattergrams were quantitated by determining the deviations fromYSI at various glucose ranges. The following results were obtained.

TABLE 6 Accuracy of MPX System from Recovery Data MPX Measurement C.V.for Range**(%) Wavelength Time (sec.) *S.D.(mg/dl) 0-50 50-250 250-450Single 20 ±5.6 7.2 14.5 — Single 30 ±6.9 7.1 8.8 10.2 Dual 20 ±2.3 5.312.8 — Dual 30 ±2.19 5.5 5.8  8.4 *= Standard Deviation **= These areinter-method covariances Note that The dual wavelength system gaveresults that ranged 30% lower than the single wavelength system. Thesingle wavelength system, from 0-50 mg/dl, showed a Standard Deviationof ±6-7 mg/dl whereas the Standard Deviation for a dual wavelengthmeasurement was only ±2.2 mg/dl. The cutoff for a 30-second MPXmeasurement is 250 mg/dl. For the 50-250 mg/dl range both the 20- and30-second measurements gave similar inter-method covariances(approximately 7% for single wavelength, 5.5% for dual wavelength).However, in the 250-450 mg/dl range inter-method covariances more thandouble for the 20-second reading to 14.5% for the single and 12.8% forthe dual wavelength. The 30-second reading was unusable above 450 mg/dlfor both the single and dual wavelength measurement (covariances of10.2% and 8.4%).

In conclusion, the two MPX systems gave optimum quantitation in the0-450 mg/dl range.

-   -   1. MPX System—30 Second Dual Wavelength: This dual wavelength        system gave a 30-second measurement time with a 95% confidence        limit (defined as the probability of a measurement being within        ±2 Standard Deviation of the YSI reading) of 11.3% covariance        for the range from 50-450 mg/dl (Table 7) and ±4.4 mg/dl        (Standard Deviation) for 0-50 mg/dl.    -   2. MPX System—30/20 Second Dual Wavelength: This dual wavelength        system gave a 20-second measurement time in the 0-250 mg/dl        range and a 30-second time for the 250-450 range. The 95%        confidence limits are nearly identical to the MPX 30 Second Dual        Wavelength system (Table 7), 11.1% covariance for 50-450 mg/dl        and ±4.6 mg/dl (Standard Deviation) for 0-50 mg/dl.

TABLE 7 Comparison of 95% Confidence Limits for the MPX System,GlucoScan Plus and Accu-Check bG**** Reagent Strips Measuring Range MPXSingle Wavelength MPX Dual Wavelength mg/dl 20 sec. 30 sec. 20 sec. 30sec.  0‥50 11.2 mg/dl 13.8 mg/dl  4.6 mg/dl  4.4 mg/dl 50-250 14.4 14.210.6 11.0 250-450  — 17.6 — 11.6 77-405 GlucoScan Plus (DrexlerClinical) 15.9% 77-405 Accu-Chek bG (Drexler Clinical) 10.7% 50-450 MPXSystem 20/30 Sec. Dual Hybrid 11.1% 50-450 MPX System 30 Sec. DualWavelength 11.3 ****Confidence limits for MPX were from the YSI. Theconfidence limits for GlucoScan Plus and Accu-Chek bG were from theregression equation vs. YSI which eliminates bias due to smalldifferences in calibration.

EXAMPLE III

Stability

Most of the bench-scale work carried out in optimizing stability wascompleted using hand-dipped 0.8μ Posidyne™ membrane disks. The specificdye/enzyme formulation was set forth previously.

-   -   1. Room Temperature Stability: This study attempted to chart any        change in response of the 0.8μ Posidyne™ membrane reagent stored        at 18° C.-20° C. over silica gel desiccant. After 2.5 months        there was no noticeable change as measured by the response of a        room temperature sample vs. the response of a sample stored at        5° C. Each measurement represented a glucose range of 0-450        mg/dl.    -   2. Stability at 37° C.: Stability study using the same reagent        as the room temperature study was carried out. The differences        in glucose values of reagent stressed at 37° C. vs. room        temperature reagent, for strips stressed with and without        adhesive, was plotted over time. Although the data was noisy,        due to the poor reproducibility of handmade strips, the        stability was excellent for strips whether they were stressed        with or without adhesive.    -   3. Stability at 56° C.: Eight 5-day to 6-day stability studies        were carried out using different preparations of a similar        formulation on disk membrane (Table 8). For the low glucose test        level (80-100 mg/dl) the average glucose value dropped upon        stressing by 3.4% with the highest drop being 9.55%. At the high        test level (280-320 mg/dl) the glucose reading declined by an        average of 3.4%, the largest decline being 10.0%.

TABLE 8 Stability of pH = 4.0, .8μ Posidyne ™ Disk Reagent FormulationStressed for 5 Days to 6 Days at 56° C. % Difference (56° C. vs. RoomTemperature Sample) Sample No. YSI (80-100 mg/dl) YSI (280-320 mg/dl)FJ22B −6.25 +5.4 FJ27A −4.0 −5.14 FJ28B −2.4 −5.3 FJ30H −9.55 −10.0FJ31C +4.43 −1.24 FJ36 −3.2 −8.5 FJ48B* −3.0 0.0 GH48A* −3.0 −2.5Average of 8 −3.4 −3.4 *These two samples contained twice the normalconcentration of enzyme and dye.

A study of the 56° C. stressing of this membrane over a 19-day periodshowed no major difference for strips stressed with or without adhesive.In both cases the 19-day decline in glucose value was less than 15% atlow test levels (80-100 mg/dl) and also at 300 mg/dl.

Another 56° C. study using hand-dipped 0.8μ Posidyne™ membrane withtwice the normal concentration of enzyme and dye was completed. Twoseparate preparations of the same formulation were made up and thestability measured over a 14-day period. The average results of the twostudies were compared. Changes were within ±10% over the 14-day periodat both the high and low glucose test level.

EXAMPLE IV

Sample Size

The sample size requirements for the MPX System are demonstrated inTable 9.

TABLE 9 Effect of Sample Size on MPX System Measurements Sample Size(μl) Dual Wavelength Single Wavelength YSI = 56 3 41 50 39 31 40 31 4230 19 30 4 44 49 49 49 48 41 45 44 45 44 5 54 48 49 51 50 50 49 48 49 4910 48 48 50 47 48 54 53 56 55 54 20 49 49 49 50 49 55 57 58 60 58 YSI =360 3 301 260 276 286 280 274 232 244 260 252 4 383 378 367 341 367 361356 342 318 344 5 398 402 382 370 388 378 387 366 351 370 10 364 362 378368 368 356 358 379 369 366 20 375 370 380 378 376 380 382 389 385 384

The volumes reported in the table were transferred to the reagent pad 10shown in FIG. 1 using a micropipet. When blood from a finger stick isapplied to a strip the total sample cannot be transferred. Therefore,the volumes reported here do not represent the total sample size neededto be squeezed from the finger for the analysis. A 3 μl sample is theminimum necessary completely cover the reagent pad circle. This does notprovide enough sample to completely saturate the reagent pad and the MPXSystem, whether single or dual wavelength, gives low results. A 4 μlsample barely saturates the reagent pad, while a 5 μl sample is clearlyadequate. A 10 μl sample is a large shiny drop and a 20 μl sample is avery large drop and is only likely to be used when blood from a pipet isused for sampling.

At the lower glucose concentration the single wavelength result has somedependence on sample size, which is completely eliminated using the dualwavelength measurement. Although this dependence with the singlewavelength might be considered acceptable, it is clearly undesirable.

EXAMPLE V

Reproducibility

Experimental measurements described above were always run in replicate,usually 2, 3 or 4 determinations per data point. These sets have alwaysshown close agreement even for samples with extremes hematocrits orextreme oxygen levels, covariances were well below 5%. It appears,therefore, that reproducibility is very good to excellent.

The subject invention provides for many commercially or have beendescribed in the literature. The protocols are simple and require littletechnical skill and are relatively free of operator error. The assayscan be carried out rapidly. They use inexpensive and relatively harmlessreagents, important considerations for materials employed in the home.The user obtains results which can be understood and used in conjunctionwith maintenance therapy. In addition, the reagents have long shelflives, so that the results obtained will be reliable for long periods oftime. The equipment is simple and reliable and substantially automatic.

All patents and other publications specifically identified in thisspecification are indicative of the level of skill of those of ordinaryskill in the art to which this invention pertains and are hereinindividually incorporated by reference to the same extent as would occurif each reference were specifically and individually incorporated byreference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many modifications and changes can bemade thereto without departing from the spirit or scope of the inventionas defined in the following claims.

1. A regent test strip for use in an apparatus for determining a bloodglucose concentration of a sample of whole blood, the apparatusincluding optical means for detecting intensity of light reflected froma reading surface portion of the reagent test strip, the reagent teststrip comprising: a reagent pad including: a sample receiving surfaceportion for receiving an unmeasured whole blood sample; and a readingsurface portion, other than the sample receiving surface portion, fromwhich reflectance is read by the apparatus; and a rigid handle to whichthe reagent pad is attached, wherein the rigid handle is configured toprovide access to the sample receiving portion by the sample and accessto the reading surface by incident light from the optical means suchthat the reagent test strip is adapted for receiving the unmeasuredwhole blood sample while in use in the apparatus.
 2. The reagent teststrip of claim 1 further adapted for receiving the unmeasured wholeblood sample while the reading surface portion is in register with theoptical means of the apparatus.
 3. The reagent test strip of claim 2,wherein the reading surface portion and the sample receiving surfaceportion are on different surfaces of the reagent test strip.
 4. Thereagent test strip of claim 3, wherein at least a portion of the surfacewith the reading surface portion thereon is substantially reflective. 5.The reagent test strip of claim 1 adapted for impregnation of a reagenttherein that reacts with blood glucose to cause a change in thereflectance at the reading surface portion.
 6. The reagent test strip ofclaim 1 adapted for allowing a portion of the unmeasured whole bloodsample to travel from the sample receiving surface portion to thereading surface portion.
 7. A reagent test strip for use in an apparatusfor determining a blood glucose concentration of a sample of wholeblood, the apparatus including optical means for detecting intensity oflight reflected from a reading surface portion of the reagent teststrip, the reagent test strip comprising: a reagent pad with a samplereceiving surface portion for receiving an unmeasured whole blood sampleand a reading surface portion, other than the sample receiving surfaceportion, from which reflectance is read by the apparatus; and a rigidhandle to which the reagent pad is attached, wherein the rigid handleincludes a hole therethrough configured to provide access to one of thesample receiving portion by the sample and the reading surface byincident light from the optical means such that the reagent test stripis adapted for receiving the unmeasured whole blood sample while in usein the apparatus.
 8. The reagent test strip of claim 7, wherein thereagent pad comprises pores of a size sufficient to exclude red bloodcells.
 9. A reagent test strip for use in an apparatus for determiningan analyte concentration of a biological fluid sample, the apparatusincluding optical means for detecting intensity of light reflected froma reading surface portion of the reagent test strip, the reagent teststrip comprising: a reagent pad including: a sample receiving surfaceportion for receiving an unmeasured biological fluid sample; and areading surface portion, other than the sample receiving surfaceportion, from which reflectance is read by the apparatus withoutremoving excess biological fluid sample from the sample receivingsurface portion; and a rigid handle to which the reagent pad isattached, wherein the rigid handle is configured to provide access tothe sample receiving portion by the sample and access to the readingsurface by incident light from the optical means such that the reagenttest strip is adapted for receiving the unmeasured biological fluidsample while in use in the apparatus.